CN104568553A - Tissue optical clearing agent and application thereof - Google Patents
Tissue optical clearing agent and application thereof Download PDFInfo
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- CN104568553A CN104568553A CN201410841350.0A CN201410841350A CN104568553A CN 104568553 A CN104568553 A CN 104568553A CN 201410841350 A CN201410841350 A CN 201410841350A CN 104568553 A CN104568553 A CN 104568553A
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Abstract
The invention provides a tissue optical clearing agent and an application thereof. The tissue optical clearing agent comprises 30-70 wt% of fructose, 2-10 wt% of octyl phenoxy poly ethoxy, 5-25 wt% of N,N,N',N'-tetra (2-hydroxypropyl) ethylene diamine and the balance of water. The tissue optical clearing agent does not damage the spatial structure of collagen in tissue, a secondary harmonic signal diagram or a fluorescent signal diagram of collagen of the tissue can be obtained, and the tissue optical clearing agent can greatly increase an imaging depth.
Description
Technical field
The present invention relates to one and organize optical transparency agent and application thereof, in particular to a kind of can maintain tissue collagen protein steric structural organize optical transparency agent and application thereof, belong to Photobiology field.
Background technology
Along with the development of contemporary optics imaging technique and the appearance of various labelling technique, high-resolution obtains mechanics of biological tissue function information becomes possibility, but the high scattering properties of biological tissue seriously constrains the application of optical image technology in biomedical sector.Multi-photon micro-imaging technique excites chromophore by adopting near infrared light, obtains fluorescence or harmonic signal, improves the degree of depth of imaging, but for the larger biological tissue of scattering, its penetration depth is still very limited.
The biotissue optical clearing technology of rising in recent years, highly to ooze by introducing in biological tissue, the chemical reagent of high refraction effectively can reduce the scattering of biological tissue, greatly improve the degree of depth of imaging.The up-to-date classical way adopted in prior art comprises: (1), method based on the serial dehydration of tetrahydrofuran, it biological tissue to be immersed in respectively in the tetrahydrofuran solution of gradient (aqueous solution of the tetrahydrofuran as 50%, 70%, 80%, 100%) 1 hour, then be immersed in respectively in methylene chloride and benzyl ether and (be respectively 45 minutes and be greater than 1 hour), make tissue become transparent; (2), use the method for urea liquid, it is in urea liquid biological tissue being immersed in 8M about one month, makes tissue become transparent; (3), based on the method for water and gel monomers conjunctive tissue electrophoresis, it is that 1-3 days is first fixed with water and gel monomers in biological tissue, then biological tissue is placed on electrophoresis 5-8 days in lauryl sodium sulfate, makes tissue become transparent.
In addition, US6472216 B1 discloses a kind of optical transparency agent, and it is selected from the group be made up of following material: dimethyl sulfoxide (DMSO), amidotrizoic acid, ethylenediamine tetraacetic acid, aminoglucose, β-nicotinamide-adenine dinucleotide phosphate salt, Sodium Amidotrizoate, polyglycol and combination thereof.CN 102749231 A discloses the agent of a kind of bone tissue optical transparency, it comprises dimethyl sulfoxide (DMSO), sodium bicarbonate, and in following substances at least two kinds: PEG-4000, gaultherolin, ethylenediamine tetraacetic acid, Sodium Diatrizoate, glycerine, glucose, neopelex, sorbierite and lauryl alcohol, after this optical transparency agent acts on bone tissue, it can be made to become optical transparency, and can imaging depth be improved.CN 103263678A discloses the agent of a kind of sufficient lift skin tissue optical transparency, it comprises fructose, ethanol, water, and in following substances at least three kinds: PEG-4000, sorbierite, glucose, propylene glycol, glycerine, neopelex, dimethyl sulfoxide (DMSO), thiophene ketone, azone, peppermint oil and vaseline, after the optical transparency agent of the program acts on sufficient lift skin tissue, tissue can be made to become optical transparency, can imaging depth be improved.Disclose in WO2013191274 A1 and use fructose soln can make embryo, forebrain transparence.
Although prior art provides the agent of Various Tissues optical transparency, but these technology are mainly based on the ultimate principle that collagen dissociates at present, the space structure of collagen can be caused to be destroyed, endogenous signal weakens and even disappears, the extreme influence effect of collagen optical second harmonic imaging.In addition, the optical transparency agent disclosed in prior art and application process length consuming time thereof; The composition more complicated of optical transparency agent.
Summary of the invention
One object of the present invention is that exploitation one organizes optical transparency agent, and it effectively can not only reduce biological tissue scatters at short notice, makes transparency of organization, can also maintain the space structure of collagen to greatest extent, thus makes endogenous optical strength unaffected; In addition, said composition can also make imaging depth be greatly improved.
Another object of the present invention, is to provide the described application organizing optical transparency agent.
For realizing the object of the invention, on the one hand, the invention provides one and organize optical transparency agent, in its general assembly (TW) for 100%, it comprises fructose, the Value 3608 of 2 ~ 10wt%, the N of 5 ~ 25wt% of 30 ~ 70wt%, N, N', N'-tetra-water of (2-hydroxypropyl) ethylenediamine and surplus.
According to the specific embodiment of the present invention, the content of each component in optical transparency agent of organizing of the present invention can according to the source of different biological tissue, age, body weight and changing, alternatively, organize in optical transparency agent of the present invention, in its general assembly (TW) for 100%, it comprises the fructose of 40 ~ 70wt%, the Value 3608 of 2 ~ 5wt%, the N of 5 ~ 10wt%, N, N', N'-tetra-water of (2-hydroxypropyl) ethylenediamine and surplus.
According to the specific embodiment of the present invention, of the present inventionly organize in optical transparency agent, in its general assembly (TW) for 100%, it is fructose, the Value 3608 of 5wt%, the N of 10wt% of 50wt%, N, N', N'-tetra-water of (2-hydroxypropyl) ethylenediamine and surplus; Or it is fructose, the Value 3608 of 10wt%, the N of 25wt% of 30wt%, N, N', N'-tetra-water of (2-hydroxypropyl) ethylenediamine and surplus; Or it is fructose, the Value 3608 of 2wt%, the N of 5wt% of 70wt%, N, N', N'-tetra-water of (2-hydroxypropyl) ethylenediamine and surplus.
Value 3608 (octylphenylpolyethylene glycol) described in the present invention, its general triton x-100 by name (triton X-100) is commercial products, buy by Reagent Company such as J & K, Alfa or Sigma-Aldrich and obtain, it has following structural formula:
Wherein, n is 9 ~ 10.
Triton x-100 in the present invention is generally chemically pure reagent, also directly can buy and use its aqueous products, is counted in the present composition by the consumption of water in wherein aqueous solution.
Of the present inventionly organize optical transparency agent to be heated to 40 ~ 60 DEG C to dissolve 2 ~ 3 hours by wherein each component simply being mixed rear water-bath, until solution to become clarification bright.
Of the present inventionly organize optical transparency agent to make transparency of organization, the homogenization of organization internal refractive index can be made, reduce tissue scatter, make tissue become transparent, for the biological information obtaining tissue provides a kind of new method.Therefore, of the present inventionly organize optical transparency agent can substitute optical transparency agent of the prior art to be applied in Angiography.Of the present inventionly organize optical transparency agent to have to make transparency of organization short time consumption short, easy and simple to handle, can make the feature of transparency of organization in about 10 ~ 30min, comparatively use the methods such as gradient tetrahydrofuran solution, urea liquid, the time shortens greatly; In addition, the composition of optical transparency agent of organizing of the present invention is simple, is only the aqueous solution of three kinds of compositions.
Therefore, on the other hand, the invention provides described optical transparency agent of organizing and make the application in transparency of organization.
According to the specific embodiment of the present invention, in application of the present invention, transparency of organization is wherein made to be second harmonic signal figure for obtaining this tissue and/or fluorescence signal.The second harmonic signal figure of the collagen mainly in this tissue or fluorescence signal.
Second harmonic figure during why the present invention can organize, mainly organize optical transparency agent can not produce destruction to the space structure of the collagen of tissue, the collagen etc. in such as, collagen in corium, the trabecular network in eyeball, arterial blood tube wall outer membrane because of of the present invention.Of the present inventionly organize each component acting in conjunction in optical transparency agent just can produce the effect not destroying collagen space structure, it is very little to the destruction of collagen in biological tissue, almost can ignore, the endogenous optical signalling of biological tissue is comparatively strong, and image quality is better.The present invention confirms that alone fructose soln can make the collagen in tissue dissociate equally.
According to the specific embodiment of the present invention, in application of the present invention, wherein, the described transparency of organization that makes makes eyeball transparent.The second harmonic signal figure of the trabecular network of the second harmonic signal figure measured in eyeball mainly in eyeball.Prior art is difficult to the second harmonic figure obtaining eyeball trabecular network, mainly destroy because optical transparency agent of the prior art can produce it on the one hand, on the other hand, the very large and trabecular network degree of depth relatively dark (degree of depth is usually at about 150 ~ 200 μm) of the cornea of the top of trabecular network and sclerotic scatter is.Of the present invention organize optical transparency agent can solution cornea and the scattering of sclera height while, keep the integrality of collagenous fibres, imaging depth can reach more than 150 μm, in a specific embodiment of the present invention, present invention obtains the second harmonic figure of eyeball 170 μm of degree of depth, show of the present inventionly to organize optical transparency agent greatly to deepen imaging depth.
According to the specific embodiment of the present invention, in application of the present invention, wherein, the described transparency of organization that makes makes skin transparent.In an embodiment of the present invention, the present invention determines the fluorescence signal figure of blood vessel under the second harmonic signal figure of skin and skin, blood vessel under skin is generally positioned at lower about 420 μm of skin, shows further of the present inventionly to organize optical transparency agent greatly to deepen imaging depth.
On the other hand, present invention also offers a kind of method making transparency of organization, the method comprises: biological tissue is immersed in described organizing in optical transparency agent and processes under room temperature, make transparency of organization.Wherein, described room temperature refers to 0 ~ 40 DEG C.In the specific embodiment of the present invention, after biological specimen can being fixed in the paraformaldehyde of 4%, rinse with PBS, then the biological sample fixed is immersed in that massfraction is 30% fructose, 10% song draw logical, 25% entprol to be configured to aqueous solution or massfraction be 70% fructose, 2% song draws logical, 5% entprol to be configured in aqueous solution, and tissue can be made to become transparent.
In method of the present invention, described in be organized in and organize the processing time in optical transparency agent to be 10 ~ 30 minutes, compared to prior art, greatly can shorten and make skin, brain, eyeball, muscle etc. be rich in the biological tissue of the collagenous fibres transparent time.
In sum, compared to optical transparency agent of the prior art, optical transparency agent of the present invention has technique effect useful as follows:
(1), of the present inventionly organizing optical transparency agent when making transparency of organization, the space structure of collagen can be maintained to greatest extent, thus make endogenous optical strength unaffected, the optical second harmonic signal of tissue or fluorescence signal are not weakened.Underlying tissue structure function information is obtained to the imaging of endogenous optical signalling and has great meaning.
(2), of the present inventionly organize optical transparency agent that imaging depth is greatly improved.
(3), use composition of the present invention that the time of transparency of organization is shortened greatly, easy and simple to handle.
Accompanying drawing explanation
Figure 1A is the second harmonic signal figure of the skin that embodiment 1 obtains;
Figure 1B is the blood vessel fluorescence signal figure under the skin that obtains of embodiment 1;
Fig. 1 C is the second harmonic signal figure of the skin that comparative example 1 obtains;
Fig. 2 is the comparison diagram that embodiment 2 eyeball adopts before and after optical transparency agent process;
Fig. 3 is the second harmonic signal figure of embodiment 2 eyeball at 0,30,60,90,120,150 μm of depth;
Fig. 4 is the second harmonic signal figure of embodiment 3 eyeball at 170 μm of depths.
Embodiment
In order to there be understanding clearly to technical characteristic of the present invention, object and beneficial effect, now in conjunction with instantiation and accompanying drawing, following detailed description is carried out to technical scheme of the present invention, these examples should be understood and be only not used in for illustration of the present invention and limit the scope of the invention.
Embodiment 1
After the amobarbital 2ml of SD rat (200g) lumbar injection 1%, cervical dislocation is put to death, with depilatory cream, the hair of rat is removed, with the skin taking off back after the skin of physiological saline cleaning rat with scissors, subcutaneous fat is removed clean with scalpel.Be soaked in massfraction after being cleaned up by rat skin with phosphate buffer and be respectively the fructose of 30%, Value 3608 (the Sigma-Aldrich reagent of 10%, n=9 ~ 10) and 25%N, N, N', in the optical transparency agent aqueous solution of N'-tetra-(2-hydroxypropyl) ethylenediamine composition, after 15-20 minute, skin becomes transparent.Transparent good skin is flattened, obtains the second harmonic signal figure (wherein excitation wavelength be 900nm, reception wavelength be 360 ~ 540nm) of skin depth about 120 μm of places with two-photon fluorescence microscope, obtain Figure 1A.Obtain dermovascular fluorescence signal figure (wherein excitation wavelength is 780nm, and reception wavelength is 360 ~ 540nm) with two-photon fluorescence microscope, obtain Figure 1B.As shown in Figure 1A, can find out that the collagenous fibres of corium are high-visible one by one, collagen does not dissolve completely.Can find out from Figure 1B transparent after can to see the fluorescence signal of veins beneath the skin through skin, this degree of depth is at about 420 μm, and super Two Photon Fluorescence far away is in the penetration depth of normal skin.
Comparative example 1
After the amobarbital 2ml of SD rat (200g) lumbar injection 1%, cervical dislocation is put to death, with depilatory cream, the hair of rat is removed, with the skin taking off back after the skin of physiological saline cleaning rat with scissors, subcutaneous fat is removed clean with scalpel.Being soaked in massfraction after being cleaned up by rat skin with phosphate buffer is in the fructose soln of 75%, 8 as a child skin become transparent.With two-photon fluorescence microscope obtain skin depth about 120 μm place (wherein excitation wavelength is 900nm, reception wavelength is 360 ~ 540nm), obtain Fig. 1 C, can find out that collagenous fibres signal is invisible as shown in Figure 1 C, collagen has dissolved completely.
Embodiment 2
After the amobarbital 2ml of SD rat (200g) lumbar injection 1%, cervical dislocation is put to death, with tweezers and scissors careful by complete for eyeball taking-up, after blood residual on phosphate buffer cleaning eyeball, the paraformaldehyde being positioned over 4% fixes 24h, rinses after taking-up with phosphate buffer.Ready eyeball is immersed in massfraction and is respectively the fructose of 70%, Value 3608 (the Sigma-Aldrich reagent of 2%, n=9 ~ 10) and 5%N, N, N', in the optical transparency agent aqueous solution of N'-tetra-(2-hydroxypropyl) ethylenediamine composition, after 15 ~ 20 minutes, eyeball becomes transparent, as shown in Figure 2, wherein in Fig. 2, left figure is eyeball before treatment, and right figure is the transparent eyeball after process.(wherein excitation wavelength is 900nm at the second harmonic signal figure at different depth place to obtain eyeball with two-photon fluorescence microscope, reception wavelength is 360 ~ 540nm), as shown in Figure 3, wherein, Fig. 3 is the second harmonic signal figure of 0,30,60,90,120 and 150 μm of degree of depth, and as can be seen from Figure 3 second harmonic signal is high-visible.
Embodiment 3
After the amobarbital 2ml of SD rat (200g) lumbar injection 1%, cervical dislocation is put to death, with tweezers and scissors careful by complete for eyeball taking-up, after blood residual on phosphate buffer cleaning eyeball, the paraformaldehyde being positioned over 4% fixes 24h, rinses after taking-up with phosphate buffer.Ready eyeball is immersed in massfraction and is respectively the fructose of 50%, Value 3608 (the Sigma-Aldrich reagent of 5%, n=9 ~ 10) and 10%N, N, N', in the optical transparency agent aqueous solution of N'-tetra-(2-hydroxypropyl) ethylenediamine composition, after 15 ~ 20 minutes, eyeball becomes transparent, (wherein excitation wavelength is 900nm at the second harmonic signal figure of 170 μm of depths to obtain eyeball with two-photon fluorescence microscope, reception wavelength is 360 ~ 540nm), obtain Fig. 4.As can be seen from Figure 4 eyeball is high-visible at the second harmonic signal at 170 μm of places.The imaging importing of the different depth adopting said method to obtain can be obtained the three-dimensional information of trabecular network, for rebuilding of trabecular network provides good foundation.
In sum, when can find out that the present invention organizes optical transparency agent can make transparency of organization, the space structure of collagen can be maintained to greatest extent, make endogenous optical strength unaffected, the optical second harmonic signal of collagen is not weakened.Imaging depth have also been obtained great raising.
Claims (10)
1. organize an optical transparency agent, in its general assembly (TW) for 100%, it comprises fructose, the Value 3608 of 2 ~ 10wt%, the N of 5 ~ 25wt% of 30 ~ 70wt%, N, N', N'-tetra-water of (2-hydroxypropyl) ethylenediamine and surplus.
2. according to claim 1ly organize optical transparency agent, wherein, in its general assembly (TW) for 100%, it comprises fructose, the Value 3608 of 2 ~ 5wt%, the N of 5 ~ 10wt% of 40 ~ 70wt%, N, N', N'-tetra-water of (2-hydroxypropyl) ethylenediamine and surplus.
3. according to claim 1ly organize optical transparency agent, in its general assembly (TW) for 100%, it is the fructose of 50wt%, the Value 3608 of 5wt%, the N of 10wt%, N, N', N'-tetra-water of (2-hydroxypropyl) ethylenediamine and surplus; Or it is fructose, the Value 3608 of 10wt%, the N of 25wt% of 30wt%, N, N', N'-tetra-water of (2-hydroxypropyl) ethylenediamine and surplus; Or it is fructose, the Value 3608 of 2wt%, the N of 5wt% of 70wt%, N, N', N'-tetra-water of (2-hydroxypropyl) ethylenediamine and surplus.
4. the optical transparency agent of organizing according to any one of claims 1 to 3 makes the application in transparency of organization.
5. application according to claim 4, wherein makes transparency of organization be second harmonic signal figure for obtaining this tissue and/or fluorescence signal figure.
6. application according to claim 5, wherein, the described transparency of organization that makes makes eyeball transparent.
7. application according to claim 5, wherein, the described transparency of organization that makes makes skin transparent.
8. make a method for transparency of organization, the method comprises:
Biological tissue is immersed in organize in optical transparency agent described in claims 1 to 3 and processes under room temperature, make transparency of organization.
9. method according to claim 8, wherein, described in be organized in and organize the processing time in optical transparency agent to be 10 ~ 30 minutes.
10. method according to claim 8 or claim 9, wherein, described in be organized as skin, brain, eyeball or muscle.
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Cited By (9)
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| CN105606573A (en) * | 2015-12-22 | 2016-05-25 | 深圳先进技术研究院 | Rapid intraoperative pathological diagnosis system and method |
| CN106289915A (en) * | 2016-09-30 | 2017-01-04 | 中国科学院自动化研究所 | Water displacement composition, dewatering and preparation method for biological organization sample |
| CN106901865A (en) * | 2017-01-06 | 2017-06-30 | 华中科技大学 | A kind of skull light transparent processing method and its application |
| CN107132101A (en) * | 2017-03-28 | 2017-09-05 | 中国科学院深圳先进技术研究院 | One kind tissue light clarifier and its preparation method and application |
| CN107621462A (en) * | 2016-07-13 | 2018-01-23 | 王志伟 | A kind of transparency of organization liquid SUT and its preparation and application |
| WO2020019670A1 (en) * | 2018-07-25 | 2020-01-30 | 中国科学院合肥物质科学研究院 | Treating liquid composition, kit, and biological organ transparentizing and immune labeling method |
| CN112504794A (en) * | 2020-11-17 | 2021-03-16 | 深圳先进技术研究院 | Tissue clearing reagent and preparation method and application thereof |
| CN113324821A (en) * | 2021-06-01 | 2021-08-31 | 华中科技大学苏州脑空间信息研究院 | Method for quickly staining biological sample and acquiring three-dimensional data |
| CN113324819A (en) * | 2021-04-19 | 2021-08-31 | 任政宇 | Composite accelerator for biological tissue transparentization and preparation method and application thereof |
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| CN105606573A (en) * | 2015-12-22 | 2016-05-25 | 深圳先进技术研究院 | Rapid intraoperative pathological diagnosis system and method |
| CN107621462B (en) * | 2016-07-13 | 2020-03-31 | 王志伟 | Tissue clearing liquid SUT and preparation and application thereof |
| CN107621462A (en) * | 2016-07-13 | 2018-01-23 | 王志伟 | A kind of transparency of organization liquid SUT and its preparation and application |
| CN106289915A (en) * | 2016-09-30 | 2017-01-04 | 中国科学院自动化研究所 | Water displacement composition, dewatering and preparation method for biological organization sample |
| CN106901865B (en) * | 2017-01-06 | 2019-10-01 | 华中科技大学 | A kind of skull light transparent processing method and its application |
| CN106901865A (en) * | 2017-01-06 | 2017-06-30 | 华中科技大学 | A kind of skull light transparent processing method and its application |
| CN107132101A (en) * | 2017-03-28 | 2017-09-05 | 中国科学院深圳先进技术研究院 | One kind tissue light clarifier and its preparation method and application |
| CN107132101B (en) * | 2017-03-28 | 2019-10-11 | 中国科学院深圳先进技术研究院 | A kind of tissue light clarifier and its preparation method and application |
| WO2020019670A1 (en) * | 2018-07-25 | 2020-01-30 | 中国科学院合肥物质科学研究院 | Treating liquid composition, kit, and biological organ transparentizing and immune labeling method |
| CN112504794A (en) * | 2020-11-17 | 2021-03-16 | 深圳先进技术研究院 | Tissue clearing reagent and preparation method and application thereof |
| CN113324819A (en) * | 2021-04-19 | 2021-08-31 | 任政宇 | Composite accelerator for biological tissue transparentization and preparation method and application thereof |
| CN113324819B (en) * | 2021-04-19 | 2023-04-21 | 任政宇 | Composite accelerator for transparentizing biological tissues and preparation method and application thereof |
| CN113324821A (en) * | 2021-06-01 | 2021-08-31 | 华中科技大学苏州脑空间信息研究院 | Method for quickly staining biological sample and acquiring three-dimensional data |
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